The present invention relates to electrofocusing the buffers. More particularly, the present invention is concerned with electrofocusing in amphoteric or non-amphoteric buffers, and with the achieving of stability with time for a natural pH gradient.
The concept of pH gradient electrophoresis has been previously considered to include the formation of a natural pH gradient due to the attainment of the isoelectric state by amphoteric compounds of a wide variety of isoelectric points. In this view of isoelectric focusing, subsequent to a relatively short transient state, in which the ampholytes approach their isoelectric positions within the pH gradient, an "equilibrium" state of long duration ensues. The stable pH gradient may then serve to focus proteins at their isoelectric positions.
The foregoing isoelectric theory has been unable to explain three facts; (1) The instability of pH gradients formed with Ampholine as a function of time, with simultaneous migration of first the basic, then the neutral, then the acid carrier ampholytes into the cathode chamber. Ampholine (or Servalyt under various trademarks) is the trade name for commercial amphoteric buffers comprised of synthetic mixtures of branched aliphatic polyamine carboxylates or sulfates.
This deterioration has been shown independently in polyacrylamide gels and in sucrose density gradients. It was not due to electroendosmosis; (2) The maintenance of a small, but finite current at the "steady-state"; and (3) The isoelectric focusing of nucleic acids.
These problems led to a consideration as to whether the pH gradient formed in focusing was not possibly identical to that formed within a stack. Therefore the buffers customarily used in polyacrylamidegel electrophoresis (PAGE) in multiphasic buffer systems (multiphasic zone electrophoresis, MZE) were tested for their ability, when mixed together at random, to yield stable pH gradients under the conditions of isoelectric focusing (IF) except for the absence of Ampholine. The success of this test led to testing of the suitability of nonamphoteric buffers as well. These experiments, indicative of the nonisoelectric nature of pH gradient formation in isoelectric focusing, are described in detail hereinafter.